The present invention is related to a method for detecting and quantifying specific DNA or RNA by using an isometric primer extension (iPE) method.
Conventional methods for detecting and quantifying special sequences of nucleic acids such as DNA and RNA include southern blotting, northern analysis, and RNase protection assays, and Polymerase Chain Reaction (PCR), among other methods. However, if the detection of a specific RNA species in a sample is considered, Northern blotting and RNase protection assay present limitations in efficiency, labor intensiveness, accuracy, high cost, sensitivity, greater RNA sample requirement, specialized equipment, and a large amount of biohazardous and radioisotopic waste material that are generated. In particular, both Northern blotting and RNase protection assay require 2-3 days for completion of the analyses. In addition, Northern blotting requires running a RNA gel, transferring the RNA to a solid support, preparing a probe, and carrying out a hybridization reaction. The sensitivity requirement is a 5 xcexcg sample for adequate sensitivity. Northern blot is based on the principle of hybridization between the target and the probe nucleic acid. Moreover, the cost per reaction is fairly high, as is the amount of biohazardous and radioisotopic waste material that are generated.
Similarly, RNase protection assay requires 2-3 days to obtain the appropriate results. The experimental procedure requires preparing template DNA, preparing RNA probe, carrying out hybridization reaction, enzyme digestion reaction, and running a gel. In order to obtain good results, a 1 xcexcg sample of the target RNA is required. The principle behind RNase protection assay is a combination of hybridization and enzyme digestion reactions. As in the Northern blotting method, it is expensive to carry out this reaction. Furthermore, biohazardous and radioisotopic waste products are generated in high amounts. A chart comparing the various factors associated with Northern blotting, RNase protection assay and the multiple primer extension method of the present invention is set forth in Table 1.
U.S. Pat. No. 5,846,710 discloses using a primer extension technique to screen for variant DNA molecules. However, this patent does not disclose detecting a target DNA or RNA in a sample.
U.S. Pat. No. 5,994,079 discloses forming an RNA/DNA hybrid by annealing a DNA primer to a specific RNA and extending the primer by using reverse transcriptase. The hybrid is detected by an antibody specific for the RNA/DNA hybrid. However, this patent does not disclose detecting a target DNA or RNA in a sample as in the present invention.
It is recognized that there is a need in the art for a nucleic acid detection method that is simple, costs less time, is sensitive, cost effective and has a low adverse environmental impact. The present invention as described hereinbelow meets all of these needs.
The present invention has met the hereinbefore described need.
The present invention is directed to a method for detecting or quantifying a target nucleic acid in a sample comprising:
(a) preparing a primer or primers specifically matched to a predetermined position of the target nucleic acid;
(b) annealing the primer or primers from (a) with the target nucleic acid under high stringency conditions to obtain a primer-nucleic acid duplex at the predetermined position of the target nucleic acid;
(c) mixing the primer-nucleic acid duplex from (b) with a mixture comprising:
(1) one or two or three types of free non-terminator nucleotides and at least one type of non-terminator nucleotide that is optionally labeled with a detectable marker, and
(2) with or without a type of terminator nucleotide that is different from the one or two or three types of non-terminator nucleotides in (1);
(d) performing the primer extension by enzymatic or chemical reaction in an appropriate buffer; and either
(e) detecting or quantifying the amount of labeling signal on the primer extended nucleotides, or
(f) detecting or quantifying the amount of extended primers by mass spectrometry.
In the above method, the primer can be a nucleic acid primer, an oligodeoxyribonucleotide, an oligoribonucleotide, or a copolymer of deoxyribonucleic acid and ribonucleic acid. The nucleic acid of interest can be a deoxyribonucleic acid, a ribonucleic acid, or a copolymer of deoxyribonucleic acid and ribonucleic acid.
In a preferred embodiment, the method may comprise using a mixture comprising a combination of non-terminator and terminator nucleotides as follows:
(a) dATP, dCTP, dGTP, ddTTP or ddUTP,
(b) dATP, dCTP, dTTP or dUTP, ddGTP,
(c) dATP, dGTP, dTTP or dUTP, ddCTP,
(d) dCTP, dGTP, dTTP, or dUTP, ddATP,
(e) dATP, dCTP, dGTP,
(f) dATP, dCTP, dTTP or dUTP,
(g) dATP, dGTP, dTTP or dUTP, or
(h) dCTP, dGTP, dTTP or dUTP.
The method of the invention may use at least one non-terminator nucleotide that is labeled with a detectable marker. The detectable marker may comprise an enzyme or protein moiety, radioactive isotope, a fluorescent moiety, or a chemical group such as biotin. Moreover, the detecting or quantifying method step may be carried out by mass spectrometry.
Some of the enzymes used in the primer extension reaction of the invention include a template-dependent enzyme such as E. coli DNA polymerase I or the xe2x80x9cKlenow fragmentxe2x80x9d thereof, T4 DNA polymerase, T7 DNA polymerase, T. aquaticus DNA polymerase, a retroviral reverse transcriptase, or a combination thereof.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.